Article pubs.acs.org/biochemistry
Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells Justin D. Cohen, Samuel Thompson, and Alice Y. Ting* Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States S Supporting Information *
ABSTRACT: Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter’s ligase (W37VLplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, E20G/W37TLplA, that ligates Pacific Blue as efficiently as W37VLplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.
P
robe incorporation mediated by enzymes (PRIME) is a method to tag recombinant proteins in living cells with chemical probes. The method utilizes mutants of E. coli lipoic acid ligase (LplA), whose natural function is to ligate lipoic acid onto acceptor proteins involved in oxidative metabolism. 1 Instead of lipoic acid, LplA mutants catalyze the covalent attachment of unnatural chemical probes, such as 7hydroxycoumarin,2 an aryl azide,3 or an alkyl azide,4 onto recombinant proteins fused to a 13-amino acid recognition sequence called LAP (LplA acceptor peptide).5 The advantages of PRIME in comparison to other protein labeling methods are the small tag size, compatibility with the interior of living cells, and high labeling specificity.6 In previous studies, uptake of the unnatural substrate by LplA was achieved by mutation of a “gatekeeper” residue, W37, at the end of the lipoic acid binding pocket (Figure 1B). Enlarging this pocket, for example by a W37 → V mutation, allows LplA to accept structures much larger than lipoic acid, such as the blue fluorophore 7-hydroxycoumarin (HC)2 (Figure 1A, top). In the course of our screening, however, we discovered several structures that are not accepted by W37 point mutants. One of the most interesting examples is Pacific Blue (PB), 7 a fluorophore that differs from HC only in the two fluorine atoms at C6 and C8 of the coumarin ring (Figure 1A, bottom). Because of these two electron-withdrawing fluorines, PB has a reduced 7-hydroxyl pKa of 3.7, compared to 7.5 for HC,7 and is therefore fully anionic and fluorescent at physiological pH (7.4) © 2011 American Chemical Society
as well as endosomal pH (5.5−6.5). In contrast, only ∼50% of HC is in the anionic and fluorescent form at pH 7.4, and it is mostly protonated and hence nonfluorescent in acidic endosomes. We hypothesized that PB is not recognized by HC’s ligase, W37V LplA, and other W37 point mutants because its negative charge clashes with the mostly hydrophobic binding pocket of LplA.8 In addition, near the W37 gatekeeper residue at the end of the lipoic acid binding tunnel is a negatively charged side chain, E20, that may electrostatically repel PB8 (Figure 1B). E20 could play a steric role as well, since a previous alanine scan in the lipoate binding pocket identified E20A as one of two mutants (along with W37A) with any detectable ligation activity for an aryl azide probe.3 The goal of this work was to use PB as a model compound to explore strategies for engineering new LplA activity, such as recognition of anionic substrates, beyond point mutations at W37. A PB ligase is also a useful alternative to HC ligase for studying proteins in acidic cellular compartments, where HC fluorescence is very low. By performing in-vitro screens using a panel of E20 and W37 single and double mutants, we discovered that E20G/W37TLplA ligates PB with comparable Received: July 6, 2011 Revised: August 20, 2011 Published: August 23, 2011 8221
dx.doi.org/10.1021/bi201037r | Biochemistry 2011, 50, 8221−8225
Biochemistry
Article
In-Vitro Screening of LplA Mutants (Figure 2A). Ligation reactions were assembled as follows for Figure 2A: 2 μM purified LplA mutant, 150 μM synthetic LAP peptide (GFEIDKVWYDLDA; synthesized by the Tufts Peptide Synthesis Core Facility), 5 mM ATP, 500 μM fluorophore probe, 5 mM magnesium acetate, and 25 mM Na2HPO4 pH 7.2 in a total volume of 25 μL. Reactions were incubated for 12 h at 30 °C. LplA mutant/probe combinations giving high activity under these conditions were then reassayed with 10-fold lower probe (50 μM) for 2 h. Product formation was analyzed by ultraperformance liquid chromatography (UPLC) on a Waters Acquity instrument using a reverse-phase BEH C18 column 1.7 μM (1.0 × 50 mm) with inline mass spectroscopy. Chromatograms were recorded at 210 nm. A gradient of 30−70% (acetonitrile + 0.05% trifluoroacetic acid) in (water with 0.1% trifluoroacetic acid) over 0.78 min was used. Further in-Vitro Screening of Top Five LplA Double Mutants (Figure 2B,C). Reactions for the top five LplA double mutants were assembled as above, but with 500 μM probe and a reaction time of 45 min. Reactions were quenched with EDTA to a final concentration of 100 mM. Product formation was analyzed on a Varian Prostar HPLC using a reverse-phase C18 Microsorb-MV 100 column (250 × 4.6 mm). Chromatograms were recorded at 210 nm. We used a 10 min gradient of 30−60% acetonitrile in water with 0.1% trifluoroacetic acid under 1 mL/min flow rate. Percent conversions were calculated by dividing the product peak area by the sum of (product + starting material) peak areas. Michaelis−Menten Kinetic Assay. The Michaelis−Menten curve shown in Figure S4 was generated as previously described.2 Reaction conditions were as follows: 2 μM E20G/W37T LplA, 600 μM synthetic LAP peptide, 2 mM magnesium acetate, and 25 mM Na2HPO4 pH 7.2. Mammalian Cell Culture and Imaging. HEK and HeLa cells were cultured in growth media consisting of Minimum Essential Medium (MEM, Cellgro) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories). Cells were maintained at 37 °C under 5% CO2. For imaging, HEK cells were grown on glass coverslips pretreated with 50 μg/mL fibronectin (Millipore) to increase their adherence. Cells were imaged in Dulbecco’s Phosphate Buffered Saline (DPBS) at room temperature. The images in Figures 3 and 4 were collected on a Zeiss AxioObserver.Z1 microscope with a 40× oil-immersion objective and 2.5× Optovar, equipped with a Yokogawa spinning disk confocal head containing a Quadband notch dichroic mirror (405/488/568/647 nm). Pacific Blue/coumarin (405 nm laser excitation, 445/40 emission filter), YFP (491 nm laser excitation, 528/38 emission filter), Alexa Fluor 568 (561 nm laser excitation, 617/73 emission filter), and DIC images were collected using Slidebook software (Intelligent Imaging Innovations). Images were acquired for 100 ms to 1 s using a Cascade II:512 camera. Fluorescence images in each experiment were normalized to the same intensity range. Cell Surface Labeling. HEK cells were transfected with 200 ng of LAP4.2-LDLR-pcDNA4 and 100 ng of H2B-YFP cotransfection marker plasmid, per 0.95 cm2 at ∼70% confluency, using Lipofectamine 2000 (Invitrogen). Fifteen hours after transfection, the growth media was removed, and the cells were washed three times with DPBS. The cells were
Figure 1. Engineering a Pacific Blue (PB) ligase. (A) Fluorophore ligations catalyzed by mutants of lipoic acid ligase (LplA). The top row shows ligation of 7-hydroxycoumarin (HC) by W37VLplA onto a LAP (LplA acceptor peptide)5 fusion protein, demonstrated in previous work.2 The bottom row shows ligation of PB by E20G/W37TLplA, demonstrated in this work. (B) Cut-away view of wild-type LplA in complex with lipoyl-AMP ester, the intermediate of the natural ligation reaction. Adapted from PDB ID 3A7R.8 W37 and E20 side chains are highlighted. (C) Modeled structure of E20G/W37TLplA in complex with PB-AMP ester. The PB-AMP conformation was energetically minimized using Avogadro.13
kinetics to W37VLplA ligation of HC (Figure 1A). We demonstrated the utility of our PB ligase for in-vitro, cell surface, and intracellular site-specific protein labeling.
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EXPERIMENTAL PROCEDURES Plasmids. The LplA-pYFJ16 plasmid was used for bacterial expression of LplA.2 The LplA-pcDNA3 plasmid was used for mammalian expression of LplA.2 For mammalian expression of LAP fusion proteins, LAP-YFP-NLS-pcDNA3, LAP4.2-neurexin-1βpNICE, and vimentin-LAP in Clontech vector were used and have been described. 2,9 The LAP sequence used was GFEIDKVWYDLDA. For some constructs (neurexin and LDL receptor), an alternative peptide sequence called LAP4.2 was used instead (GFEIDKVWHDFPA).5 LAP4.2-LDLRpcDNA4 was generated from HA-LDLR-pcDNA410 by a two-stage QuikChange to insert the LAP4.2 sequence and was a gift from Daniel Liu (MIT). The nuclear YFP transfection marker was H2B-YFP and has been described. 11 All mutants were prepared by QuikChange mutagenesis. LplA Expression and Purification. LplA mutants were expressed in BL21 E. coli and purified by His6-nickel affinity chromatography as previously described.2 8222
dx.doi.org/10.1021/bi201037r | Biochemistry 2011, 50, 8221−8225
Biochemistry
Article
labeled by applying 100 μM Pacific Blue or hydroxycoumarin probe, 2 μM ligase, 1 mM ATP, and 5 mM Mg(OAc)2 in DPBS at room temperature for 40 min. Cells were then washed three times with DPBS and either imaged immediately or incubated at 37 °C for an additional 30 min to allow receptor internalization prior to imaging. Intracellular Protein Labeling. HEK cells were transfected at ∼70% confluency with 200 ng of LAP-YFP-NLSpcDNA3 and 50 ng of FLAG-E20G/W37TLplA-pcDNA3 per 0.95 cm2 using Lipofectamine 2000 (Invitrogen). Fifteen hours after transfection, the growth media was removed, and the cells were washed three times with serum-free MEM. The cells were labeled by applying 20 μM PB3-AM2 in serum-free MEM at 37 °C for 20 min. The cells were then washed three times with fresh MEM. Excess probe was removed by changing the media several times over 40 min. To visualize LplA expression levels, cells were fixed using 3.7% formaldehyde in PBS pH 7.4 for 10 min, followed by methanol at −20 °C for 5 min. Fixed cells were washed with DPBS and then blocked overnight with blocking buffer (3% BSA in DPBS with 0.1% Tween-20). Anti-FLAG M2 antibody (Sigma) was added at a 1:300 dilution in blocking buffer for 1 h at room temperature. Cells were then washed three times with DPBS before treatment with a 1:300 dilution of goat antimouse antibody conjugated to Alexa Fluor 568 (Invitrogen) in blocking buffer for 1 h at room temperature. Cells were washed three times with DPBS prior to imaging. For labeling of vimentin-LAP (Figure 4B), HeLa cells were transfected with 250 ng of vimentin-LAP-Clontech, 50 ng of FLAG-E20G/W37TLplA-pcDNA3, and 100 ng of H2B-YFP transfection marker per 0.95 cm2 using Lipofectamine 2000. Labeling was performed as above, with an extended 60 min washout period to remove excess probe. Cells were then imaged live in DPBS. We note that, compared to intracellular labeling with hydroxycoumarin, labeling with PB3 generally requires longer washout times, up to 60 min in some cases. Shorter wash times result in higher PB background in all cells.
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RESULTS Screening for a Pacific Blue Ligase. On the basis of the LplA crystal structure (Figure 1B),8 we decided to focus our engineering efforts on the W37 and E20 positions. We started with a preliminary screen of 19 W37 point mutants and 14 E20 point mutants, against four probe structures (Figure S1). These four structures, shown in Figure 2A, are two Pacific Blue probes with shorter (n = 3) and longer (n = 4) linkers (PB3 and PB4) and two analogous 7-hydroxycoumarin probes (HC3 and HC4). Some Pacific Blue (PB) ligation product was detected after a 12 h reaction with W37T, V, I, and A LplA mutants (Figure S1), so we decided to introduce these mutations into our next screen. Note that the activity of the best point mutant, W37T LplA, which gave ∼50% conversion to PB ligation product after 12 h, is too slow for practical utility. For E20, none of the tested point mutants gave product with any of the four probes after 12 h. Nevertheless, in our next screen, we included E20 mutations to the smaller, neutral side chains Gly, Ala, and Ser. Our next library consisted of 7 single mutants (four at W37 and three at E20) and their 12 crossed double mutants, shown in Figure 2A. Screening was performed using 500 μM probe in an overnight reaction. Any ligase/probe combination with high activity under these conditions was reassayed using 50 μM
Figure 2. Screening of LplA mutants for Pacific Blue ligation activity. (A) Relative product conversions measured for 19 LplA single and double mutants with two hydroxycoumarin (HC) probes and two Pacific Blue (PB) probes. HC3 and PB3 have n = 3 linkers, and HC4 and PB4 have n = 4 linkers. To generate these grids, ligation reactions were performed under both forcing conditions (12 h, 500 μM probe) and milder conditions (2 h, 50 μM probe) and analyzed by ultraperformance liquid chromatography, as described in the Experimental Procedures. Sample traces are shown in Figure S2. The activity grid was generated with the following tiers: no activity,
